Name: intrauterine telemetry to measure mouse contractile pressure in vivo
Uploaded: 2015-04-06T15:00:00.000+00:00
Duration: 7 min 3 s
Description: Watch this Scientific Journal Video about Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo at JoVE.com

This work was supported by funding from the March of Dimes FY12-133 (S.K.E.) and the National Institutes of Health R01HD037831 (S.K.E.). We would like to thank Dr. Deborah Frank for critical review of the manuscript.

NOTE: All animal procedures complied with guidelines for the care and use of animals set forth by the National Institutes of Health. All protocols were approved by the Animal Studies Committee at Washington University in St. Louis. 
NOTE: Specific materials and equipment for this protocol are listed Materials and Equipment table.
1. Timed Breeding of Female Mice
<ol>
	<li>Breed adult females between two and six months of age in a 2 hr time window with males. Specific breeding hr are not needed as long as they are kept consistent for the entirety of the experiment.</li>
	<li>Confirm pregnancy by the presence of a copulatory plug. Designate the day and hr on which the plug is first seen as day post-coitus 0 (dpc0). Remember the specific hour when starting pressure recording.</li>
	<li>Perform surgeries on dpc8&#8211;13. The exact dpc will depend on the mouse strain.</li>
	<li>To determine if a mouse is pregnant before surgery, perform ultrasound imaging of the abdomen with a 40 MHz linear array probe coupled to a imaging system before administering anesthesia. This will allow visualization of gestational sacs. If an ultrasound system is not available, use the weight of the mouse. 
	NOTE: A weight gain of at least 2 g between dpc0 and dpc8 and distension of the abdomen is a good indicator of pregnancy.</li>
</ol>
2. Telemetry Surgery Prep
<ol>
	<li>Use an autoclave to sterilize instruments, gauze, cotton swabs, surgical drapes, and pipette tips within 24 hr before surgery.</li>
	<li>Clean surgery surface with 70% ethanol.</li>
	<li>Turn on heating pad (specialized for rodents) and bead sterilizer.</li>
</ol>
3. Telemetry Surgery
<ol>
	<li>For anesthesia, use 4-5% isoflurane with an oxygen flow rate of 1-2 L/min.</li>
	<li>Wait until the mouse shows signs of anesthetization and then inject subcutaneously with buprenorphine (analgesic:&#160;0.1 ml per 20 g).</li>
	<li>Shave the abdomen of the mouse from the bottom of the ribs down to the bladder.</li>
	<li>Bring the mouse to the surgery table on pre-warmed heating pad, and cover the mouse&#8217;s eyes with ophthalmic ointment&#160;to prevent drying.</li>
	<li>Begin sterile technique. Put on sterile gloves and keep the wrapper as a sterile surface on which to place the iodine and ethanol scrubs.</li>
	<li>Remove the telemeter from the package or saline and use sterile forceps to place it on sterile gauze.
	<ol>
		<li>Use the syringe (provided by the telemeter supplier) to fill the catheter tip with gel.&#160; Gently squeeze the catheter creating enough negative pressure to draw the gel into the catheter.</li>
	</ol>
	</li>
	<li>Place a sterile tip on the pipette. Pipette 2 &#181;l of surgical glue and set the pipette on a sterile surface.</li>
	<li>To clean the surgical site, swab the area with iodine and then with 70% ethanol.</li>
	<li>Cut a hole in the surgical drape just large enough for the area needed for the incision and place over the mouse.</li>
	<li>Perform a foot pinch (pedal response) to ensure the mouse is completely anesthetized before making an incision.</li>
	<li>Make a small vertical midline incision. Next, make a similar incision through the underlying body wall muscle.</li>
	<li>Gently pull the uterus out of the body cavity and locate the uterine horn containing the most pups that are viable.
	<ol>
		<li>Optional - For addition of miRNA, inject viral vector into muscle layer of uterus before insertion of telemeter.</li>
	</ol>
	</li>
	<li>Make a small incision at the tip of the uterine horn with 3 mm cutting edge spring scissors.</li>
	<li>Thread the catheter, without squeezing, between the uterine wall and fetal sacs. Insert the catheter past several sacs to reduce its accidental removal. Make sure the fetal sacs are not disturbed by the insertion.</li>
	<li>Pipette the surgical glue to the site of telemeter insertion to adhere the catheter to the uterine horn and prevent the catheter from sliding out of the uterine horn. Wait a few seconds for the glue to become rigid, and then carefully place the uterus back inside the body cavity.</li>
	<li>Place the telemeter in the body cavity on the opposite side of the catheter insertion site.</li>
	<li>Suture the body cavity closed with 6.0 absorbable sutures. First, do a purse stitch and then three to four individual stitches.</li>
	<li>Suture the skin by using the same technique, but with 6-0 non-absorbable suture.</li>
	<li>Swab dibucaine (topical analgesic) ointment over the incision site.</li>
	<li>Inject 0.3-0.4 ml of sterile saline subcutaneously to help rehydrate the animal.</li>
	<li>Monitor the animal after surgery until it is fully awake. Do not place the mouse back in its cage until it is able to freely hold its head above the table as certain types of bedding can suffocate a heavily anesthetized mouse. For a mouse that has been anesthetized for 30-45 min, one should expect the mouse to be fully awake by 1 hr and 15 min.</li>
	<li>If performing additional surgeries, wipe instruments with 70% ethanol and bead sterilize before reuse.</li>
</ol>
4. Post-surgery Recovery
<ol>
	<li>Monitor daily post-surgery progress in a log. Catalog weight gain and loss and watch for lethargy, bleeding, or incomplete closure of the surgical incision.
	<ol>
		<li>Feed the mouse softened mouse chow (~the consistency of applesauce) or specialized recovery diet food.</li>
		<li>Examine the sutures and make sure they are not pulled out, but try not to handle the mouse excessively.</li>
	</ol>
	</li>
</ol>
5. Telemetry Recording
<ol>
	<li>On the day of interest (at least a two to three days after the mouse has recovered from surgery), place a magnet near the mouse to turn on the telemeter. Put the mouse cage on the receiver. The light on the receiver should turn on if the telemeter is activated.</li>
	<li>Calibrate brand new telemeters with manufacturer specific calibrations provided. Reused telemeter calibration specifications will be kept in software.</li>
	<li>Within the software, start recording by right clicking on &#8220;animal&#8221; and click &#8220;start sampling, continuous,&#8221; which allows sampling at rates of up to 500 Hz. Make sure the software is set to save AND trace. 
	NOTE: When the sampling mouse icon turns green, pressures are being recorded. Disturbing the mouse during delivery can interrupt or alter labor, so observe cautiously.</li>
	<li>After delivery or time period of interest, stop recording by right clicking on &#8220;animal&#8221; and selecting &#8220;stop recordings, all&#8221;. Use a magnet to turn off telemeter to save battery life, as the telemeter battery will typically only last for 1.5 months of continuous recording.</li>
	<li>Euthanize the mouse by CO2 overdose and recover the telemeter. Pups are euthanized by decapitation.&#160;</li>
</ol>
6. Telemeter Sterilization
<ol>
	<li>Place the telemeter in a solution of 1% enzyme detergent in deionized water overnight at room temperature.</li>
	<li>Rinse the telemeter in deionized water and remove all tissue and glue, being careful not to squeeze the catheter.</li>
	<li>Incubate the telemeter in 2% glutaraldehyde for 24 hr at room temperature to sterilize.</li>
	<li>Rinse the telemeter with sterile saline and store it in sterile saline until next implantation or to be sent back to manufacturer to be refurbished. Telemeters should be refurbished if tissue is located inside the catheter after sterilization. Typically telemeters can be used 4-5 times before needing to be refurbished.</li>
</ol>

<ol>
	<li>Slattery, M. M., Morrison, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preterm+delivery.">Preterm delivery.</a> Lancet. 360, 1489-1497 (2002).</li><li>Goldenberg, R. L., Culhane, J. F., Iams, J. D., Romero, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+causes+of+preterm+birth.">Epidemiology and causes of preterm birth.</a> Lancet. 371, 75-84 (2008).</li><li>Keelan, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+inhibition+of+inflammatory+pathways+for+the+prevention+of+preterm+birth.">Pharmacological inhibition of inflammatory pathways for the prevention of preterm birth.</a> Journal Of Reproductive Immunology. 88, 176-184 (2011).</li><li>Voltolini, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+spontaneous+preterm+birth:+from+underlying+mechanisms+to+predictive+and+preventive+interventions.">Understanding spontaneous preterm birth: from underlying mechanisms to predictive and preventive interventions.</a> Reproductive Sciences. 20, 1274-1292 (2013).</li><li>Ratajczak, C. K., Muglia, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+parturition+biology+from+genetically+altered+mice.">Insights into parturition biology from genetically altered mice.</a> Pediatric research. 64, 581-589 (2008).</li><li>Rajakumar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+HIF-1+alpha,+HIF-2+alpha,+membrane+and+soluble+VEGF+receptor-1+proteins+are+not+increased+in+normotensive+pregnancies+complicated+by+late-onset+intrauterine+growth+restriction.">Placental HIF-1 alpha, HIF-2 alpha, membrane and soluble VEGF receptor-1 proteins are not increased in normotensive pregnancies complicated by late-onset intrauterine growth restriction.</a> American Journal Of Physiology. Regulatory, Integrative And Comparative Physiology. 293, (2007).</li><li>Whitesall, S. E., Hoff, J. B., Vollmer, A. P., D'Alecy, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+simultaneous+measurement+of+mouse+systolic+arterial+blood+pressure+by+radiotelemetry+and+tail-cuff+methods.+American+journal+of+physiology.">Comparison of simultaneous measurement of mouse systolic arterial blood pressure by radiotelemetry and tail-cuff methods. American journal of physiology.</a> Heart And Circulatory Physiology. 286, H2408-H2415 (2004).</li><li>Mackay, L. B., Shi, L. B., Maul, H. .., Maner, W. L., Garfield, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+bilateral+pelvic+neurectomy+on+cervical+ripening+in+pregnant+rats.">The effect of bilateral pelvic neurectomy on cervical ripening in pregnant rats.</a> Journal Of Perinatal Medicine. 37, 263-269 (2009).</li><li>Pierce, S. L., Kutschke, W., Cabeza, R., England, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+intrauterine+pressure+by+telemetry:+a+new+approach+for+studying+parturition+in+mouse+models.">In vivo measurement of intrauterine pressure by telemetry: a new approach for studying parturition in mouse models.</a> Physiol Genomics. 42, 310-316 (2010).</li><li>Imamura, T., Luedke, C. E., Vogt, S. K., Muglia, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxytocin+modulates+the+onset+of+murine+parturition+by+competing+ovarian+and+uterine+effects.">Oxytocin modulates the onset of murine parturition by competing ovarian and uterine effects.</a> American Journal Of Physiology. Regulatory, Integrative And Comparative Physiology. 279, R1061-R1067 (2000).</li><li>McCloskey, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inwardly+rectifying+K++channel+KIR7.1+controls+uterine+excitability+throughout+pregnancy.">The inwardly rectifying K+ channel KIR7.1 controls uterine excitability throughout pregnancy.</a> EMBO Mol Med. 6 (9), 1161-1174 (2014).</li></ol>

The authors have nothing to disclose.

Investigations of myometrial contractility have relied on ex vivo measurements of muscle tension. This methodology can be useful for early-stage testing of newly developed uterotonic drugs that cannot be administered to live animals. However, in vivo approaches are necessary to longitudinally understand the progression of labor. The in vivo measurements serve several purposes. First, they enable capture of a complete picture of changes in uterine contractile pattern over the duration of pregnancy. Second, they eliminate the bias of ex vivo recordings that require uterine tissue to be mounted either vertically or horizontally, limiting our understanding of the complete role of the circular and longitudinal muscle layer of the uterus. Third, they allow recording of uterine pressure in the endogenous hormonal environment, thus making it possible to more accurately study parturition in mouse models. Finally, they allow for more translational investigations between mouse models and humans as uterine telemetry more closely resembles measurements taken in pregnant women with intrauterine pressure catheters.
The methods for mouse uterine telemetry are easy to learn, but difficult to master. Investigators with previous mouse surgery experience may achieve success faster, but this shouldn&#8217;t deter labs from using this valuable technique. The original studies using mouse uterine telemetry were done by a novice with great success after a few tries, so labs should be prepared for the first couple of surgeries to cause the dam to lose all fetuses. Here are a few pointers to keep in mind. As with any surgery, the shortest duration is the best for the animal. A quick surgery will limit dehydration and the need for additional doses of anesthesia. If the researcher is having a difficult time finishing the procedure before the mouse wakes up, isoflurane can be used to keep the mouse anesthetized longer. We have also found that the use of isoflurane increases the number of viable pups and is preferred over ketamine. The smallest incision possible, just slightly bigger than the body of the telemeter, will reduce the chance of the wound gaping later, decrease time needed to suture, and improve mouse recovery. With a small abdominal incision, pulling the uterus outside of the body cavity will allow for better visibility during catheter insertion. When reusing a telemeter, factor in the previous curve of the catheter when threading it down the uterine horn. Allowing the surgical glue to harden completely before placing the uterine horn back inside the body cavity will help keep the catheter in place and prevent the glue from sticking to the intestines. The telemeter body can damage the liver if placed too high, and the mouse&#8217;s abdomen is capable of expanding extensively, so do not hesitate to place the telemeter lower in the body. These methods will have to be customized to fit the needs of different labs and different mouse models, but the advantages of uterine telemetry far outweigh the obstacles.
There are some limitations that should be considered when implementing this methodology. One of the main challenges that have to be overcome is the degree of fetal loss following implantation of the transmitter. In the intrauterine telemetry initial report9, all transgenic SK3T/T and wild type mice had some degree of fetal loss in one or both uterine horns regardless if that horn was chosen for telemeter placement. Optimization of the choice of day during pregnancy when the telemeter was surgically inserted improved our rate of successful term pregnancies, but a full complement of pups was never obtained. Notably, the optimal day of surgery for SK3T/T mice differed from that in wild type, with better reproductive outcomes noted at dpc12-13 for SK3T/T mice versus dpc8-9 for WT mice, suggesting that the surgery protocol may need to be modified for each mouse model of interest. Our data indicated that the number of pups birthed did not correlate with overall intrauterine pressure. Fetal outcomes did improve with improved surgical skills by the experimenter, and others who have implemented this methodology in their laboratory have noted that fetal outcomes improved with the use of isoflurane instead of ketamine. It is expected that as more laboratories use this methodology, the transmitter technology and intrauterine analysis software will become more refined. This will undoubtedly advance the usefulness of this technology in studies of the mechanisms that contribute to labor.

Preterm birth is the leading cause of perinatal morbidity and mortality in developed countries; it is responsible for 50% of perinatal morbidity and 75% of perinatal mortality1,2. Preterm labor is multifaceted and can be idiopathic. Although much research has emerged on the molecular and electrical pathways transforming the myometrium from a quiescent tissue into a contractile one, the exact pathophysiology of preterm labor remains elusive. Endocrine, inflammatory, and gene regulation have all been linked to preterm labor3,4. However, ethical concerns limit the ability to conduct research on the mechanisms of preterm labor in humans.
Given these limitations, many researchers have turned to mice as a model system with which to study the physiology of parturition. Mice have short gestations lasting approximately three weeks and can be easily genetically manipulated. Additionally, multiple genetic mouse models have been developed to determine the signaling pathways that are essential for labor5. Despite key differences between mouse and human parturition, mice and humans share many of the same mechanisms that are essential for labor, including intrauterine inflammation and infection6. Thus, mice serve as an invaluable tool for examining uterine activity. To date, the gold standard to measure uterine contractility in mice has been ex vivo isometric tension recordings; however, this is limited to one gestational time point per experiment and requires removal of the uterus from its physiological environment. Another important factor is the large number of animals required for these investigations. Lastly, this methodology does not allow for longitudinal studies examining induction of labor and preterm pathophysiology.
Recent advancements made in radiometric telemetry devices used to study arterial pressure changes7 in mice and intrauterine pressure in rats8 begged the question to whether the same technology could be used to study changes in intrauterine pressure in mice during pregnancy. After initial troubleshooting, a method was successfully developed to measure labor induction and progression in a mouse. This in vivo approach can measure the transition from the low-pressure state of the quiescent uterus to the high-pressure state indicative of forceful labor contractions. This method has also been able to detect significant pressure differences between transgenic mice with compromised parturition and wild type mice, demonstrating the physiological impact of gene expression9. This protocol provides a real-time, in vivo method to study mouse uterine pressure during pregnancy.

<table><tbody><tr><td>Surgical Glue</td><td>3M Vetbond</td><td>1469SB</td><td>Tissue Adhesive--stored in Drierite</td></tr><tr><td>Absorbable 6-0 Sutures</td><td>Ethicon</td><td>J212H</td><td>Vicryl</td></tr><tr><td>Non-absorbable 6-0 Sutures</td><td>Ethicon</td><td>8706H</td><td>Prolene</td></tr><tr><td>Water Jacket Blanket + Heating Pad</td><td>Gaymer</td><td>T/Pump PN 11184-000 Blanket-66N111CC</td><td>Specialized for rodents</td></tr><tr><td>Bead sterilizer</td><td>Keller</td><td>Z378577</td><td>Steri 250 Sterilizer</td></tr><tr><td>Disecting Microscope</td><td>Nikon</td><td>SMZ754</td><td>Fibre optic gooseneck external light source</td></tr><tr><td>Sterile Surgical Gloves--Latex&amp;nbsp;</td><td>Cardinal Health Triflex</td><td>2D7253</td><td></td></tr><tr><td>PhysioTel PA-C10 Pressure Transmitter</td><td>&amp;nbsp;Data Sciences International</td><td>270-0135-001</td><td>TA11PA-C10</td></tr><tr><td>Telemeter Reciever</td><td>&amp;nbsp;Data Sciences International</td><td>272-6001-001</td><td>RPC-1</td></tr><tr><td>Dataquest ART 4.3.2 Analysis Platinum</td><td>&amp;nbsp;Data Sciences International</td><td>271-0147-141</td><td>Analysis software</td></tr><tr><td>Diet Recovery Gel</td><td>Clear H2O</td><td>72-01-5022</td><td>Purified Soft Diet for Rodents</td></tr><tr><td>Tergazyme</td><td>Sigma-Aldrich</td><td>Z273287</td><td>Enzyme detergent</td></tr></tbody></table>

intrauterine telemetry to measure mouse contractile pressure in vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Real-time in vivo intrauterine pressures can be recorded by using a telemetric acquisition system. Pressure sampling paired with simultaneous video recording of the mouse was used to capture the exact time of each pup&#8217;s delivery and correlate delivery time to the intrauterine pressure of the mother. All the data points can then be plotted throughout the recording to generate a pressure-versus-time plot (Figure 1A). This plot shows the points during gestation when the intrauterine pressure changes as well as the difference in pressure between the dark and light cycles. Additionally, a moving average of the intrauterine pressure can be plotted (Figure 1B). This is useful to more easily visualize sustained increases in intrauterine pressure characteristic of the contractions associated with the labor process (recordings obtained during delivery have been published previously9).
Data can be further analyzed by exporting data points to analysis software and averaging data over the time period of interest. When comparing different experimental conditions, such as wild type versus transgenic mice, daily or hourly pressures from different mice can be averaged to examine contractile patterns dependent on the gene of interest. Additionally, the statistical significance of any differences can be determined. Intrauterine pressures can be examined during delivery, or a longitudinal study can be conducted to examine the entire gestational period under different conditions.
Intrauterine telemetry can be used to test in vivo responses to pharmacological agents during pregnancy. Specifically, this method allows for early drug testing prior to clinical trials. Differences in response to specific agents can be tested in transgenic mice, as well as in non-pregnant or pregnant mice. One specific pharmacological agent known to increase uterine contraction, oxytocin, can be delivered subcutaneously as previously described in a rat model10. The telemeter can be implanted at the same time as a micro-osmotic pump to deliver a steady rate of oxytocin. Differences in recordings between non-pregnant (Figure 2A) and pregnant (Figure 2B) mice with administration of oxytocin can also be examined.
Another potential application of intrauterine telemetry is looking at uterine contractions after miRNA/shRNA silencing of proteins. Mouse models are not available for all genes that are essential for parturition. Thus, gene silencing in combination with intrauterine pressure measurements can be used to investigate proteins of interest. By injecting miRNA-expressing lentiviral vectors into the myometrium during the implantation of the telemeter, uterine-specific knockdown of a gene of interest can be examined, as seen in Figure 3A. A recent study shows a specific example demonstrating that the Kir7.1 channel has an important role in uterine quiescence after using both miRNA and telemetry to examine changes to intrauterine pressure throughout gestation11. Representative tracings of scrambled miRNA (Figure 3A) or Kir7.1 miRNA (Figure 3B) show that knocking down expression of the potassium channel Kir7.1 caused intrauterine pressure to increase. These representative tracings are similar to what is seen in transgenic mouse models as previously described9.
<img alt="Figure 1" src="/files/ftp_upload/52541/52541fig1highres.jpg" /> 
Figure 1: Representative continuous intrauterine pressure recording. (A) Real-time intrauterine pressure data recorded in vivo continuously for four days (15-19 dpc). (B) Moving hourly average of data in (A). White and black bars on top of recordings indicate 12 hr light and dark cycles, respectively. (C) A zoomed in trace of mouse in vivo individual contractions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52541/52541fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52541/52541fig2highres.jpg" /> 
Figure 2: Intrauterine pressures in response to oxytocin. Representative tracings of intrauterine pressure from non-pregnant (NP) myometrium (A) and late pregnant (18 dpc) myometrium (B) exposed to 5 units/day of oxytocin for 24 hr show increases in pressure in the late pregnant versus NP mice. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52541/52541fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52541/52541fig3highres.jpg" /> 
Figure 3: Measurement of intrauterine pressure after silencing of a potassium channel by miRNA/shRNA. (A) Schematic of the placement of the telemeter and sites of injection of miRNA-expressing lentiviruses. Recent studies have shown the representative tracings of intrauterine pressure from myometrium transduced with (B) scrambled miRNA or (C) Kir7.1 miRNA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52541/52541fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo at JoVE.com

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

intrauterine-telemetry-to-measure-mouse-contractile-pressure-in-vivo

Nanyang Technological University

Research

JoVE Journal

Medicine

9.6K Views.  Washington University in St. Louis. This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy. 

Video: Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

Measuring Left Ventricular Pressure in Late Embryonic and Neonatal Mice

Blood pressure increases significantly during embryonic and postnatal development in vertebrate animals. In the mouse, blood flow is first detectable around embryonic day (E) 8.51. Systolic left ventricular (LV) pressure is 2 mmHg at E9.5 and 11 mmHg at E14.52. At these mid-embryonic stages, the LV is clearly visible through the chest wall for invasive pressure measurements because the ribs and skin are not fully developed. Between E14.5 and birth (approximately E21) imaging methods must be used to view the LV. After birth, mean arterial pressure increases from 30 - 70 mmHg from postnatal day (P) 2 - 353. Beyond P20, arterial pressure can be measured with solid-state catheters (i.e. Millar or Scisense). Before P20, these catheters are too big for developing mouse arteries and arterial pressure must be measured with custom pulled plastic catheters attached to fluid-filled pressure transducers3 or glass micropipettes attached to servo null pressure transducers4.
Our recent work has shown that the greatest increase in blood pressure occurs during the late embryonic to early postnatal period in mice5-7. This large increase in blood pressure may influence smooth muscle cell (SMC) phenotype in developing arteries and trigger important mechanotransduction events. In human disease, where the mechanical properties of developing arteries are compromised by defects in extracellular matrix proteins (i.e. Marfan's Syndrome8 and Supravalvular Aortic Stenosis9) the rapid changes in blood pressure during this period may contribute to disease phenotype and severity through alterations in mechanotransduction signals. Therefore, it is important to be able to measure blood pressure changes during late embryonic and neonatal periods in mouse models of human disease.
We describe a method for measuring LV pressure in late embryonic (E18) and early postnatal (P1 - 20) mice. A needle attached to a fluid-filled pressure transducer is inserted into the LV under ultrasound guidance. Care is taken to maintain normal cardiac function during the experimental protocol, especially for the embryonic mice. Representative data are presented and limitations of the protocol are discussed.

Measuring left ventricular pressure (LV) in embryonic and neonatal mice is described. Pressure is measured by inserting a needle connected to a fluid-filled transducer into the LV under ultrasound guidance. Care must be taken to maintain normal cardiac function during the experimental protocol.

Blood pressure increases significantly during embryonic and postnatal development in vertebrate animals. In the mouse, blood flow is first detectable ...

Bioengineering

Implantation of Radiotelemetry Transmitters Yielding Data on ECG, Heart Rate, Core Body Temperature and Activity in Free-moving Laboratory Mice

The laboratory mouse is the animal species of choice for most biomedical research, in both the academic sphere and the pharmaceutical industry. Mice are a manageable size and relatively easy to house. These factors, together with the availability of a wealth of spontaneous and experimentally induced mutants, make laboratory mice ideally suited to a wide variety of research areas.
In cardiovascular, pharmacological and toxicological research, accurate measurement of parameters relating to the circulatory system of laboratory animals is often required. Determination of heart rate, heart rate variability, and duration of PQ and QT intervals are based on electrocardiogram (ECG) recordings. However, obtaining reliable ECG curves as well as physiological data such as core body temperature in mice can be difficult using conventional measurement techniques, which require connecting sensors and lead wires to a restrained, tethered, or even anaesthetized animal. Data obtained in this fashion must be interpreted with caution, as it is well known that restraining and anesthesia can have a major artifactual influence on physiological parameters1, 2.
Radiotelemetry enables data to be collected from conscious and untethered animals. Measurements can be conducted even in freely moving animals, and without requiring the investigator to be in the proximity of the animal. Thus, known sources of artifacts are avoided, and accurate and reliable measurements are assured. This methodology also reduces interanimal variability, thus reducing the number of animals used, rendering this technology the most humane method of monitoring physiological parameters in laboratory animals3, 4. Constant advancements in data acquisition technology and implant miniaturization mean that it is now possible to record physiological parameters and locomotor activity continuously and in realtime over longer periods such as hours, days or even weeks3, 5.
Here, we describe a surgical technique for implantation of a commercially available telemetry transmitter used for continuous measurements of core body temperature, locomotor activity and biopotential (i.e. onelead ECG), from which heart rate, heart rate variability, and PQ and QT intervals can be established in freeroaming, untethered mice. We also present pre-operative procedures and protocols for post-operative intensive care and pain treatment that improve recovery, well-being and survival rates in implanted mice5, 6.

A surgical technique for implantation of commercially available telemetry transmitters used for continuous measurement of biopotential (one-lead ECG), heart rate, core body temperature and locomotor activity in freely moving mice is shown. Recommendations and protocols for post-operative care and pain relief, improving recovery, well being and survival rate are also presented.

The laboratory mouse is the animal species of choice for most biomedical research, in both the academic sphere and the pharmaceutical industry. Mice are a ...

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

Mouse Maternal Autonomic Changes from Pregnancy to Pup-Rearing

Analysis of Electrocardiograms and Behavior in Mice from Pregnancy to Lactation Period

Changes in the mother-offspring relationship are presumably accompanied by dynamic changes in the autonomic nervous system. Although temporal measurements of autonomic activity have been performed in human mothers and infants, the analysis of long-term changes remains unexplored. Mouse mothers can form social bonds with their pups and have a short period of pregnancy and lactation, which makes them useful for the examination of physiological changes from pregnancy to pup-rearing. Therefore, a telemetry system was used for several weeks to measure the changes in the autonomic nervous system and the behavior of mouse mothers. The current results showed that an electrocardiogram (ECG) could be stably recorded regardless of the movements of mothers and parturition. ECG analysis showed that the heart rate gradually decreased from pregnancy to lactation, and sympathetic activity sharply increased as the pups developed. Furthermore, the simultaneous recording of behavior and ECG in the home cage enabled us to understand the behavior-dependent influences on the ECG, thereby revealing the characteristics of autonomic nervous activity during each behavior. Thus, the present experimental method helps to understand how the physiological characteristics of mothers change from pregnancy through pup rearing, supporting the healthy development of pups.

A simultaneous recording of autonomic activity and detailed maternal behavior of mother mice from pregnancy to lactation was achieved using a telemetry system. This method helps to understand the dynamics of the physiological and behavioral characteristics in mothers from pregnancy to weaning.

Changes in the mother-offspring relationship are presumably accompanied by dynamic changes in the autonomic nervous system. Although temporal measurements ...

Behavior

Long-term Blood Pressure Measurement in Freely Moving Mice Using Telemetry

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. Indeed, research focusing on the discovery of new potential therapeutic targets using genetically altered mice requires a reliable, long-term assessment of the systemic arterial pressure variation. Currently, the gold standard for obtaining long-term measurements of blood pressure in ambulatory mice uses implantable radio-transmitters, which require&#160;artery cannulation. This technique eliminates the need for tethering, restraining, or anesthetizing the animals which introduce stress and artifacts during data sampling. However, arterial blood pressure monitoring in mice via catheterization can be rather challenging due to the small size of the arteries. Here we present a step-by-step guide to illustrate the crucial key passages for a successful subcutaneous implantation of radio-transmitters and carotid artery cannulation in mice. We also include examples of long-term blood pressure activity taken from freely moving mice after a period of post-surgery recovery. Following this procedure will allow reliable direct blood pressure recordings from multiple animals simultaneously.

The goal of this protocol is to assess systemic blood pressure in conscious freely moving mice using implantable radio-telemetry devices.

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.

Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage.

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...

Immunology and Infection

Implantation of Combined Telemetric ECG and Blood Pressure Transmitters to Determine Spontaneous Baroreflex Sensitivity in Conscious Mice

Blood pressure (BP) and heart rate (HR) are both controlled by the autonomic nervous system (ANS) and are closely intertwined due to reflex mechanisms. The baroreflex is a key homeostatic mechanism to counteract acute, short-term changes in arterial BP and to maintain BP in a relatively narrow physiological range. BP is sensed by baroreceptors located in the aortic arch and carotid sinus. When BP changes, signals are transmitted to the central nervous system and are then communicated to the parasympathetic and sympathetic branches of the autonomic nervous system to adjust HR. A rise in BP causes a reflex decrease in HR, a drop in BP causes a reflex increase in HR.
Baroreflex sensitivity (BRS) is the quantitative relationship between changes in arterial BP and corresponding changes in HR. Cardiovascular diseases are often associated with impaired baroreflex function. In various studies reduced BRS has been reported in e.g., heart failure, myocardial infarction, or coronary artery disease. 
Determination of BRS requires information from both BP and HR, which can be recorded simultaneously using telemetric devices. The surgical procedure is described beginning with the insertion of the pressure sensor into the left carotid artery and positioning of its tip in the aortic arch to monitor arterial pressure followed by the subcutaneous placement of the transmitter and ECG electrodes. We also describe postoperative intensive care and analgesic management. After a two-week period of post-surgery recovery long-term ECG and BP recordings are performed in conscious and unrestrained mice. Finally, we include examples of high-quality recordings and the analysis of spontaneous baroreceptor sensitivity using the sequence method.

The baroreflex is a heart-rate regulation mechanism by the autonomic nervous system in response to blood-pressure changes. We describe a surgical technique to implant telemetry transmitters for continuous and simultaneous measurement of electrocardiogram and blood pressure in mice. This can determine spontaneous baroreflex sensitivity, an important prognostic marker for cardiovascular disease.

Blood pressure (BP) and heart rate (HR) are both controlled by the autonomic nervous system (ANS) and are closely intertwined due to reflex mechanisms. ...

Real-Time Electrocardiogram Monitoring During Treadmill Training in Mice

Regular physical exercise is a major contributor to cardiovascular health, influencing various metabolic as well as electrophysiological processes. However, in certain cardiac diseases such as inherited arrhythmia syndromes, e.g., arrhythmogenic cardiomyopathy (ACM) or myocarditis, physical exercise may have negative effects on the heart leading to a proarrhythmogenic substrate production. Currently, the underlying molecular mechanisms of exercise-related proarrhythmogenic remodeling are largely unknown, thus it remains unclear which frequency, duration, and intensity of exercise can be considered safe in the context of disease(s).
The proposed method allows to study proarrhythmic/antiarrhythmic effects of physical exercise by combining treadmill training with real-time monitoring of the ECG. Implantable telemetry devices are used to continuously record the ECG of freely moving mice over a period of up to 3 months both at rest and during treadmill training. Data acquisition software with its analysis modules is used to analyze basic ECG parameters such as heart rate, P wave duration, PR interval, QRS interval, or QT duration at rest, during and after training. Furthermore, heart rate variability (HRV) parameters and occurrence of arrhythmias are evaluated. In brief, this manuscript describes a step-by-step approach to experimentally explore exercise induced effects on cardiac electrophysiology, including potential proarrhythmogenic remodeling in mouse models.

Electrocardiogram (ECG) is the key variable to understanding cardiac electrophysiology. Physical exercise has beneficial effects but may also be harmful in the context of cardiovascular diseases. This manuscript provides a method of recording real-time ECG during exercise, which can serve to investigate its effects on cardiac electrophysiology in mice.

Regular physical exercise is a major contributor to cardiovascular health, influencing various metabolic as well as electrophysiological processes. ...

Ex Vivo Method for Assessing the Mouse Reproductive Tract Spontaneous Motility and a MATLAB-based Uterus Motion Tracking Algorithm for Data Analysis

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. Here, we present an easy and inexpensive method that would be instrumental for testing new drugs decreasing uterine contractility. This method utilizes the unique ability of the entire mouse reproductive tract to exhibit spontaneous motility when maintained ex vivo in a Petri dish containing oxygenated Krebs buffer. This spontaneous motility resembles the wave-like myometrial activity of the human uterus, referred to as endometrial waves. To demonstrate the effectiveness of the method, we employed a well-known uterine relaxant drug, epinephrine. We demonstrate that the spontaneous motility of the entire mouse reproductive tract can be quickly and reversibly inhibited by 1 &#181;M epinephrine in this Petri dish model. Documenting the changes of uterine motility can be easily done using an ordinary smart phone or a sophisticated digital camera. We developed a MATLAB-based algorithm allowing motion tracking to quantify spontaneous uterine motility changes by measuring the rate of uterine horn movements. A major advantage of this ex vivo approach is that the reproductive tract remains intact throughout the entire experiment, preserving all intrinsic intrauterine cellular interactions. The major limitation of this approach is that up to 10-20% of uteri may exhibit no spontaneous motility. Thus far, this is the first quantitative ex vivo method for assessing spontaneous uterine motility in a Petri dish model.

Uterine contractions are important for the well-being of females. However, pathologically increased contractility may result in dysmenorrhea, especially in younger females. Here, we describe a simple ex vivo preparation allowing quick assessment of the efficacy of smooth muscle relaxants that may be used for treating dysmenorrhea.

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. ...

Biology

Continuous Telemetric In Utero Tracheal Pressure Measurements in Fetal Lambs

Normal in utero lung development and growth rely upon the expansion of airspaces and the controlled efflux of lung liquid into the amniotic space. Infants with congenital diaphragmatic hernia (CDH) also have lung hypoplasia due to occupation of the chest cavity by the stomach and bowel and, in the most severe cases, the liver. Balloon tracheal occlusion reduces the severity of lung hypoplasia in fetuses with CDH but increases the risk of premature birth. Understanding the optimal occlusion pressure and duration required to improve lung hypoplasia with tracheal occlusion is essential to improving in utero corrective treatments for CDH. The study reports a new method for continuous measurement of the intratracheal and amniotic pressures in an unoccluded and occluded fetal lamb surgical model of CDH. Time-pregnant Merino ewes underwent two recovery hysterotomies: the first at ~80 days of gestation to create the CDH, and the second at ~101 days of gestation to occlude the fetal trachea and implant an intratracheal and amniotic pressure measurement device. Lambs were delivered at ~142 days, and the pressure measurement device was removed and cleaned. The data were downloaded and filtered using a 6 h window. Transrespiratory pressure was calculated.

Continuous Telemetric In Utero Tracheal Pressure Measurements in Fetal Lambs

Optimization of occlusion pressure and duration required to improve lung hypoplasia with tracheal occlusion is essential to improve in utero corrective treatments for congenital diaphragmatic hernia (CDH). This study reports a new method for continuous measurement of the tracheal pressure in an occluded and unoccluded fetal lamb surgical model of CDH.

Normal in utero lung development and growth rely upon the expansion of airspaces and the controlled efflux of lung liquid into the amniotic space. Infants ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

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